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  • Antimycotic Effects of Octenidine and Pirtenidine

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    Antimycotic Effects of Octenidine and Pirtenidine

    Diunggah oleh

    Mohamed
    Publication

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    Ciimicrobial Chemotherapy (1989) 24, 00-00 Ly a fTERSET JACI57P 19.0989 L11 py i ot \ | s ie yusis — wi | ooev j nN Antimycotic effects of octenidine and pirtenidine | ) | x 'M. A. Ghannoum’, K. Abu Elteen’, M. Ellabib® and P, A. Whittaker? | “Department of Botany and Microbiology, Kuwait University, Kuvait; "Department of Biology, Maynooth College, Maynooth, Ireland i “The gfets of octenidine and pirteniine on yeasts (in particular Candid albicans) hgY'been studied, MIC and MCC values have been established as well as the inhibitory effects on growth, budding end germ cube formation. The drugs were how to cause extensive Teskage of cytoplasmic contents from the ells which was Cortelated with morphological and ultrastructural changes inthe yeas. Introduction ‘The increasing significance of fungal infections in man and other animals has been’ widely reported (Armstrong et al., 1975; Kichn, Edwards & Armstrong, 1980; Odds, je 1988). There are a number of factors which have contributed to this increased significance in recent years (Ghannoum, 1988; Odds, 1988). These include the wide- spread use of antibacterial antibiotics, the development and use of immunosuppressive ‘agents in the treatment of organ transplant patients and patients suffering from autoimmune or immune deficiency diseases (Hurley, de Louvois & Mulhall, 1987). The dimorphic yeast Candida albicans is without doubt the most important pathogenic fungus to have increased in significance due to these factors (Mirsky & Cuttner, 1972). ‘The problems caused by this increase in fungal pathogenesis are of greater impor- : tance because of the relative lack of antifungal agents which are both effective and free of side effects (Speller, 1980), It is consequently very important to investigate thor~ oughly any new drug with'antimycotic potential, to establish its mode of action and, clinical possibilities Ti this paper we describe some studies related (o the antimycotic effects of two drugs, ‘etenidine and pirtenidine (Figure 1) with some structural similarity to chlorhexidine, which have been developed for potential use as antibacterial mouthwashes. Although these were originally selected for their antibacterial properties, preliminary studies (Sedlock & Bailey, 1985; Whittaker & Ellabib, unpublished) established that they also exhibited antimycotic properties, Materials and methods: Drugs COvienidine hydrochovide (NAAJ110. dcanediy-t[4H-pyridny-4ytidenebis-- tctanamine) dihydrochloride] aad prtenidine [N-(I-octy-4 1} pytiinylidene) oct ttamine monohydrochlorde) (Figure 1) are new drugs developed by tering- Winthrop ‘Correspondence: Professor P. A. Whitaker, —- ' 7 00 M A. Ghannoum et af. nae a + on (oH) = N(CH) CH, - Cr | =’ 0 | Pirtenidine Ee aS — N= (CH) ON’ LO i cHICH), Octenidine cr NH CNH CNH (CH) NHC NH C NH ci i non aon NH NH NH NH . Chlorhexidine eucture of pirtenidine,octenidine and chlorhexidine. | | Figur Research Institute as antimicrobial or antiplaque agents. These drugs were gifts from Sterling-Wimhrop. Organisms Yeast species used were: C. albicans KCCC 14172, C. tropicalis KCCC 13622 and C. pseudotropiealis KCCC 13709, which were isolated from the oral cavities of patients tindergoing head and neck radiation therapy at Kuwait Cancer Control Centre, and . Iyophilyzed. ‘The isolation and identification techniques used have already been described (Ghannoum et al., 1985a, b). C. albicans ATCC 10231 which was isolated from a patient suffering from bronchomycosis and obtained as a lyophilized sample from the American Type Culture Collection, USA and Saccharomyces cerevisiae NCYC 975 were also used. All of the organisms were maintained on slopes of Sabouraud modified agar (Difco), stored at 4°C, and subcultured routinely ‘Determination of minimum inkibitory concentrations (MIC) and minimum cidal concentrations (MCC) Serial two-fold dilutions of the drugs ranging from 800 mg/l to 0-2 mg/l were prepared irom S00 ngs! ae ~ tlextrose (YNBD) and inoculated with 001 ml of afresh overnight broth culture. The tubes were then inoculated with 0-01 ml of an overnight culture and then incubated at 37°C for 24h. The MIC values were noted as the lowest concentration showing no visible growth. One loopful of the broth tubes showing no visible growth was further subeultured on to a Sabouraud agar plate to determine the MCC. The plates were incubated at 37°C for 24h and then checked for viability Efjects on growth An inoculum of C. albicans KCCC 14172 and S. cerevisiae was grown overnight at 37°C with rotary agitation in YNBD. The cells were centrifuged and resuspended in a small volume of fresh medium. This suspension was used to inoculate (10° cells/ml) 4 100m of fresh medium containing O (control), x MIC, 4x MIC and 1x MIC of the drugs. The flasks were incubated at 37°C, Samples were withdrawn at 60 min intervals, and the rate of growth of shake cultures was followed by determining the ‘optical density at 420 nm (SP6-550 Pye Unicam), Action on budding celts C. albicans KCCC 14172 and S. cerevisiae were maintained on YNBD for 48 h. The cells were centrifuged and washed three times with distilled water. Yeast cells (10?/ml) were inoculated into flasks contsining 20 mi of Eagle's medium (which supports bud formation of yeasts) with and without drugs. Flasks were incubated at 25°C on a gyratory shaker at 200 rpm. Samples were taken at intervals and scored for bud formation by counting the mean number of yeasts forming buds in every 300 yeast cals Effeets on germ tube formation ©. albicans KCCC 14172 cells grown with and without drugs were washed three times with distilled water. An inoculum from each preparation was added to tubes containing. calf serum (Gibeo) and incubated in a shaking water bath at 37°C. Samples were taken at imtervals and added to an equal volume 1% glutaraldehyde in phosphate bulfer- saline (PBS) for fixation. The number of yeasts with germ tubes was determined microscopically (Soll, Bedell & Brummel, 1981) Leakage of intracellular material Equal volumes (5 ml) of octenidine and pirtenidine solutions (O (control), 3x MIC, 4%MIC and 1 x MIC) and cell suspensions were mixed to give a final cell concentra- tion of 1 mg (wet weight)/ml and incubated at 24°C. At intervals cells were removed by centrifugation (7000 g, 5 min). Cellular exudates were determined by direct spectropho- tomettie measurement of the material absorbing at 260 nm in the supernatant. Scanning electron microscopy (SEM) C. albicans KCCC 14172 and S. cerevisiae were grown in flasks containing 100 ml ‘PNBD with ond withcitt the dines fo hae Colts at APO Or 1.32. Ki A Came eT 000 M. A. Ghannoum et af — Transmission electron microscopy (TEM) ie Calls were fixed with 2:5% (vj) glutaraldehyde in 0-1 M sodium cacodylate buffer, pH 74, at °C for 2h, Cells were placed in freshly made 2% (w/v) KMnO, solution at 4°C| for 2h, The cells were centrifuged at 4000 rpm for Smin and placed in afresh solution | ‘of KMaO, for 2h. They were washed five times with distilled water. The cells were then | placed in a solution containing 1% (w/v) patassium dichromate and 1% (w/v) uranyl | acetate for 2h at 4%C. These were washed several times with distilled water and) embedded in agar, left to set and cut into small cubes (0+5-I mm?) which were] dehydrated through an ethanol series. The 100% ethanol was replaced with propylene | oxide twice for 20 min and the sample was embedded in Epon by graded impregnation. | Sections were obtained using an ultramicrotome, counterstained with lead citrate, and observed under a JEOL 100 CX microscope. Results Inhibitory and mycocidal effects, 1C vc bet The Ss TECIF of octenidine and pirtenidine against each yeast strain tested S#fibetween 1-5 and 30mg/l. There was more variation in the mycocidal effects of the drugs — S. cerevisiae being the most sensitive, with the MCC values being the same as the MIC values. for the Candida strains MCC values were between 12-5 and 25 mg/l ‘The effects of different concentrations of the drugs on the growth curves of C. albicans KCCC 14172 and S. cerevisiae showed that in all cases growth inhibition was concentration-dependent. Figure 2 shows the growth curves obtained using pirtenidine | i | ee 6-0 Loa cet number 50 ° Time (8) | Figure 2. Effect of pirtenidine on growth of C. albicans (KCCC 14172), Experimental procedure is described in Matenals"and Methonls section, Pireniine concentration (mil) C), O (contol), 4, O75, a aaite GE ba t= CURED, A, 9 (1 RT. | i | | | | | | “eerie 3 Leakage of eytopiasni material fom C. atbicans KCCC 4172 and 5: ‘cerevisiae induced by Peemiting Octentine concentration (gi) C:abicen: W, O (contol); @, O75 Cx TxMMICh we re 1 MIQE A, 20 = 2 MICh,S. cere: 7, 0 Contes 0,075 (= Joe MIC) DTS eRe, 4.80 (= 35 MIC), and C. albicans, with exponential growth in the presence of the drug but increased doubling times. Qualitatively similar results were obtained with both drugs and with both yeast species Both octenidine and pirtenidine were shown to inhibit the extensive budding initiated on transfer of cither C, albicans KCCC 14172 or S. cerevisiae to Eagle's medium, The drugs also blocked germ ttibe formation in C, albicans on incubation in calf serum, Leakage of intracellular material Leakage of cytoplasmic material from C. albicans and S. cerevisiae cells inoculated into various octenidine concentrations is shown in Fisure 3. The deur ind. se - Antimycotic effects of ocenidine and pircnidine oon {—_____ z >} os | ee. i | | on goo | 5 j i | * oe | | o1 | Poe ne | 5 © 2025 0 60 Amtico cts ected and rte om leakage of cellular contents from the yeasts. 5. cerevisiae was more susceptible than C—- atcons. Qualtatively sina resus were obsrved using preidine, although les UVeabsorbing material was eased. ! leeiron microscopy minimal ihibtery concentration (mg) of octenidne. Control eels (Figure Mn) {xhibited a smooth-walle appearance and were spherical to ovoid in shape They ne {nthe yeas form and showedcear evidence of budding. AMter hf inebation Pipes (0) some of the cells showed evidence of weakening in the call envoge cate ination of cell colapse in some cases. These lets increased in 3 and came {ot shows) which culminated ater 24h (Figure) in extensive cell dames, hae and collapse of cel structure and extrusion of ellular contents. Qualities ventas resus were observed using either drug and also with. cerevias ‘Transmission eletron micrographs of control C. albicont elle (Figure (a) showed. the ell walls beeame iregla in suture with deformities inthe layering chet, and apparent loss of intemal cohesion. Call membranes lst thet inary aed _xtoplamic contents o-havy coagulated, giving rise to elecrondense and sheng ne retions. Similar observations were made when octeniine was, reploed ahi pitenidine, Discusion ontents. The effects observed paralel those reported for chlorhexidine (Dobson Rouchet 1987. These workers speculated on the mechanism of action for chimes | dine and concluded that coagulation of nucleoplasm was aerial event inthe eter this drug. No biochemical evidence was presented for this, however, In the ease cy Senne and pinendine we cannot as yet be cern of their primary target bot reo

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